U.S. patent number 7,749,740 [Application Number 10/923,458] was granted by the patent office on 2010-07-06 for microbial production of pyruvate and pyruvate derivatives.
This patent grant is currently assigned to University of Georgia Research Foundation, Inc.. Invention is credited to Elliot Altman, Mark A. Eiteman.
United States Patent |
7,749,740 |
Eiteman , et al. |
July 6, 2010 |
Microbial production of pyruvate and pyruvate derivatives
Abstract
Microbial production of pyruvate and metabolites derived from
pyruvate in cells exhibiting reduced pyruvate dehydrogenase
activity compared to wild-type cells. Acetate and glucose are
supplied as a carbon sources.
Inventors: |
Eiteman; Mark A. (Athens,
GA), Altman; Elliot (Athens, GA) |
Assignee: |
University of Georgia Research
Foundation, Inc. (Athens, GA)
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Family
ID: |
27760520 |
Appl.
No.: |
10/923,458 |
Filed: |
August 20, 2004 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050255572 A1 |
Nov 17, 2005 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/US03/05083 |
Feb 20, 2003 |
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60359279 |
Feb 20, 2002 |
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60402747 |
Aug 12, 2002 |
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Current U.S.
Class: |
435/136; 435/183;
536/23.2; 435/252.3; 435/190; 435/320.1; 435/116 |
Current CPC
Class: |
C12P
13/06 (20130101); C12P 7/26 (20130101); C12P
7/40 (20130101) |
Current International
Class: |
C12P
13/06 (20060101); C12N 9/00 (20060101); C12N
9/04 (20060101); C12N 1/20 (20060101); C07H
21/04 (20060101); C12N 15/00 (20060101); C12P
7/40 (20060101) |
Field of
Search: |
;435/116,136,183,190,252.3,320,320.1 ;536/23.2 |
References Cited
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10220234 |
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Aug 2007 |
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DE |
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Nov 2007 |
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DE |
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Aug 1999 |
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EP |
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WO 03/000913 |
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03/070913 |
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03/070913 |
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Aug 2003 |
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WO |
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WO 03/093488 |
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Nov 2003 |
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WO |
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Vemuri et al., "Succinate production in dual-phase Escherichia coli
fermentations depends on the time of transition from aerobic to
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|
Primary Examiner: Fronda; Christian L
Attorney, Agent or Firm: Mueting, Raasch & Gebhardt,
P.A.
Parent Case Text
This application is a continuation-in-part of International
Application No. PCT/US03/05083, filed Feb. 20, 2003, which claims
the benefit of U.S. Provisional Application No. 60/359,279 (filed
Feb. 20, 2002) and U.S. Provisional Application No. 60/402,747
(filed Aug. 12, 2002), all of which are incorporated by reference
in their entireties.
Claims
What is claimed is:
1. A method for making a metabolite comprising pyruvate or a
pyruvate derivative, the method comprising: providing a bacterial
cell exhibiting reduced activity of at least one enzyme in the
pyruvate dehydrogenase (PDH) complex of enzymes, compared to a
wild-type bacterial cell; and culturing the bacterial cell in the
presence of glucose and acetate under conditions and for a time
effective to accumulate the metabolite to a concentration of at
least about 3.3 g/L.
2. The method of claim 1 wherein the metabolite comprises pyruvate
or diacetyl.
3. The method of claim 1 wherein the bacterial cell further
exhibits added or increased NADH oxidase activity compared to a
wild-type bacterial cell.
4. The method of claim 1 wherein the metabolite comprises
alanine.
5. The method of claim 1 further comprising culturing the bacterial
cell in the presence of an additional carbon source comprising a
compound that is part of the tricarboxylic acid cycle of the
bacterial cell.
6. The method of claim 5 wherein the additional carbon source
comprises succinate.
7. The method of claim 1 wherein the metabolite comprises
pyruvate.
8. The method of claim 7 wherein pyruvate is produced in an amount
of at least about 30 g/L.
9. The method of claim 7 wherein the pyruvate yield is at least
about 0.70.
10. The method of claim 1 wherein the PDH activity in the bacterial
cell is undetectable.
11. A method for making a metabolite comprising pyruvate or a
pyruvate derivative, the method comprising: providing an E. coli
cell exhibiting reduced activity of at least one enzyme in the
pyruvate dehydrogenase (PDH) complex of enzymes, compared to a
wild-type E. coli cell; and culturing the E. coli cell in the
presence of glucose and acetate to yield the metabolite.
12. The method of claim 11 wherein the metabolite comprises
pyruvate or diacetyl.
13. The method of claim 11 wherein the E. coli cell further
exhibits added NADH oxidase activity.
14. The method of claim 11 wherein the metabolite comprises
alanine.
15. The method of claim 11 further comprising culturing the E. coli
cell in the presence of an additional carbon source comprising a
compound that is part of the tricarboxylic acid cycle of the E.
coli cell.
16. The method of claim 15 wherein the additional carbon source
comprises succinate.
17. The method of claim 11 wherein the metabolite comprises
pyruvate.
18. A method for making a metabolite comprising pyruvate or a
pyruvate derivative, the method comprising: providing a bacterial
cell exhibiting (a) reduced activity of at least one enzyme in the
pyruvate dehydrogenase (PDH) complex of enzymes compared to a
wild-type bacterial cell, and (b) reduced activity of
phosphoenolpyruvate carboxylase (PEP carboxylase) compared to a
wild-type bacterial cell; and culturing the bacterial cell in the
presence of glucose and acetate to yield the metabolite.
19. The method of claim 18 further comprising culturing the
bacterial cell in the presence of an additional carbon source
comprising a compound that is part of the tricarboxylic acid cycle
of the bacterial cell.
20. The method of claim 19 wherein the additional carbon source
comprises succinate.
21. The method of claim 19 wherein the metabolite comprises
pyruvate.
22. A method for making a metabolite comprising pyruvate or a
pyruvate derivative, the method comprising: providing a bacterial
cell exhibiting (a) reduced activity of at least one enzyme in the
pyruvate dehydrogenase (PDH) complex of enzymes compared to a
wild-type bacterial cell, and (b) added or increased NADH oxidase
activity; and culturing the bacterial cell in the presence of
glucose and acetate to yield the metabolite.
23. The method of claim 22 further comprising culturing the
bacterial cell in the presence of an additional carbon source
comprising a compound that is part of the tricarboxylic acid cycle
of the bacterial cell.
24. The method of claim 23 wherein the additional carbon source
comprises succinate.
25. The method of claim 23 wherein the metabolite comprises
pyruvate.
26. A method for making a metabolite comprising pyruvate or a
pyruvate derivative, the method comprising: providing a bacterial
cell exhibiting (a) reduced activity of at least one enzyme in the
pyruvate dehydrogenase(PDH) complex of enzymes, compared to a
wild-type bacterial cell, and (b) reduced activity of pyruvate
oxidase compared to a wild-type bacterial cell; and culturing the
bacterial cell in the presence of glucose and acetate to yield the
metabolite.
27. The method of claim 26 further comprising culturing the
bacterial cell in the presence of an additional carbon source
comprising a compound that is part of the tricarboxylic acid cycle
of the bacterial cell.
28. The method of claim 27 wherein the additional carbon source
comprises succinate.
29. The method of claim 27 wherein the metabolite comprises
pyruvate.
30. A method for making a metabolite comprising pyruvate or a
pyruvate derivative, the method comprising: providing a bacterial
cell exhibiting (a) reduced activity of at least one enzyme in the
pyruvate dehydrogenase (PDH) complex of enzymes, compared to a
wild-type bacterial cell, (b) reduced activity of
phosphoenolpyruvate carboxylase (PEP carboxylase) compared to a
wild-type bacterial cell, and (c) reduced activity of pyruvate
oxidase compared to a wild-type bacterial cell; and culturing the
bacterial cell in the presence of glucose and acetate to yield the
metabolite.
31. The method of claim 30 further comprising culturing the
bacterial cell in the presence of an additional carbon source
comprising a compound that is part of the tricarboxylic acid cycle
of the bacterial cell.
32. The method of claim 31 wherein the additional carbon source
comprises succinate.
33. The method of claim 30 wherein the metabolite comprises
pyruvate.
34. A method for making a metabolite comprising pyruvate or a
pyruvate derivative, the method comprising: providing a bacterial
cell exhibiting (a) reduced activity of at least one enzyme in the
pyruvate dehydrogenase (PDH) complex of enzymes, compared to a
wild-type bacterial cell, (b) reduced activity of
phosphoenolpyruvate carboxylase (PEP carboxylase) compared to a
wild-type bacterial cell, (c) reduced activity of pyruvate oxidase
compared to a wild-type bacterial cell, and (d) added or increased
NADH oxidase activity; and culturing the bacterial cell in the
presence of glucose and acetate to yield the metabolite.
35. The method of claim 34 further comprising culturing the
bacterial cell in the presence of an additional carbon source
comprising a compound that is part of the tricarboxylic acid cycle
of the bacterial cell.
36. The method of claim 35 wherein the additional carbon source
comprises succinate.
37. The method of claim 34 wherein the metabolite comprises
pyruvate.
38. A method for making pyruvate comprising: providing a bacterial
cell wherein the gene encoding at least one enzyme in the pyruvate
dehydrogenase (PDH) complex of enzymes is knocked out; and
culturing the bacterial cell in the presence of glucose and acetate
to yield the metabolite.
39. The method of claim 38 further comprising culturing the
bacterial cell in the presence of an additional carbon source
comprising a compound that is part of the tricarboxylic acid cycle
of the bacterial cell.
40. The method of claim 39 wherein the additional carbon source
comprises succinate.
41. The method of claim 38 wherein the bacterial cell further
exhibits reduced activity of pyruvate oxidase.
42. The method of claim 38 wherein the bacterial cell further
exhibits reduced activity of phosphoenolpyruvate carboxylase (PEP
carboxylase).
43. The method of claim 38 wherein the bacterial cell further
exhibits increased or added activity of NADH oxidase.
44. The method of claim 38 wherein the bacterial cell further
exhibits reduced activity of pyruvate oxidase and reduced activity
of PEP carboxylase.
45. The method of claim 38 further wherein the bacterial cell
further exhibits reduced activity of pyruvate oxidase, reduced
activity of PEP carboxylase, and increased or added activity of
NADH oxidase.
46. The method of claim 38 wherein the metabolite comprises
pyruvate.
47. A method for making a metabolite comprising pyruvate or a
pyruvate derivative, the method comprising: providing an E. coli
cell wherein the gene encoding at least one enzyme in the pyruvate
dehydrogenase (PDH) complex of enzymes is knocked out; and
culturing the E. coli cell in the presence of glucose and acetate
to yield the metabolite.
48. The method of claim 47 further comprising culturing the E. coli
cell in the presence of an additional carbon source comprising a
compound that is part of the tricarboxylic acid cycle of E.
coli.
49. The method of claim 47 wherein the additional carbon source
comprises succinate.
50. The method of claim 47 wherein the metabolite comprises
pyruvate.
51. A method for making alanine comprising: providing a bacterial
cell exhibiting (a) reduced activity of at least one enzyme in the
pyruvate dehydrogenase (PDH) complex of enzymes, compared to a
wild-type bacterial cell, and (b) added or increased alanine
dehydrogenase activity compared to a wild-type bacterial cell; and
culturing the bacterial cell in the presence of glucose and acetate
to yield alanine.
52. The method of claim 51 further comprising culturing the
bacterial cell in the presence of an additional carbon source
comprising a compound that is part of the tricarboxylic acid cycle
of the bacterial cell.
53. The method of claim 52 wherein the additional carbon source
comprises succinate.
54. A method for making alanine comprising: providing an E. coli
cell exhibiting (a) reduced activity of at least one enzyme in the
pyruvate dehydrogenase (PDH) complex of enzymes, compared to a
wild-type bacterial cell, and (b) added or increased alanine
dehydrogenase activity compared to a wild-type E. coli cell; and
culturing the E. coli cell in the presence of a glucose and acetate
to yield alanine.
55. The method of claim 54 further comprising culturing the E. coli
cell in the presence of an additional carbon source comprising a
compound that is part of the tricarboxylic acid cycle of E.
coli.
56. The method of claim 55 wherein the additional carbon source
comprises succinate.
57. A method for making diacetyl comprising: providing a bacterial
cell exhibiting (a) reduced activity of at least one enzyme in the
pyruvate dehydrogenase (PDH) complex of enzymes, compared to a
wild-type bacterial cell, and (b) added or increased acetolactate
synthase activity; and culturing the bacterial cell in the presence
of glucose and acetate to yield diacetyl.
58. The method of claim 57 further comprising culturing the
bacterial cell in the presence of an additional carbon source
comprising a compound that is part of the tricarboxylic acid cycle
of the bacterial cell.
59. The method of claim 58 wherein the additional carbon source
comprises succinate.
60. The method of claim 57 wherein the bacterial cell further
exhibits added or increased NADH oxidase activity.
61. A method for making diacetyl comprising: providing an E. coli
cell exhibiting (a) reduced activity of at least one enzyme in the
pyruvate dehydrogenase (PDH) complex of enzymes, compared to a
wild-type E. coli cell, and (b) added or increased acetolactate
synthase activity; and culturing the E. coli cell in the presence
of glucose and acetate to yield diacetyl.
62. The method of claim 61 further comprising culturing the E. coli
cell in the presence of an additional carbon source comprising a
compound that is part of the tricarboxylic acid cycle of E.
coli.
63. The method of claim 62 wherein the additional carbon source
comprises succinate.
64. The method of claim 61 wherein the E. coli cell further
exhibits added NADH oxidase activity.
Description
BACKGROUND OF THE INVENTION
In stating a vision for the future in "Biobased Industrial
Products, Priorities for Research and Commercialization", the
National Research Council has proposed U.S. leadership for the
global transition to biobased products. The report provides
compelling evidence for a competitively priced biobased products
industry that will eventually replace much of the petrochemical
industry. In light of this report and the desirability of reducing
U.S. reliance on foreign oil, there is an increasing interest in
generating commodity and fine chemicals from widely available U.S.
renewable resources, e.g., crops, through fermentation. In the last
few years, companies have invested hundreds of millions of dollars
in commercializing the microbial production of several
biochemicals, such as lactic acid.
Microbial fermentation processes are used to generate a wide
variety of important biochemicals such as ethanol and lysine
(markets in the billions of U.S. dollars). In order to be economic,
fermentations rely on microorganisms which have been developed by
selection or genetic means to accumulate a specific product that is
produced via metabolism. Microbial metabolic pathways are not
naturally optimal for the generation of a desired chemical, but
have instead evolved for the benefit of the organism. Metabolic
engineering is the targeted and rational alteration of metabolism,
and it involves the redirection of cellular activities to generate
a new product or generate a product at a higher rate or yield.
Pyruvate (pyruvic acid) is a three-carbon ketoacid synthesized at
the end of glycolysis. Pyruvate is an important raw material for
the production of L-tryptophan, L-tyrosine,
3,4-dihydroxyphenyl-L-alanine, and for the synthesis of many drugs
and biochemicals. Pyruvate has use in the chemical industry and
finds wide application in cosmetics. Clinical studies have found
that pyruvate can promote weight loss and fat loss, hence it is
commonly marketed in tablet form as a dietary supplement. Recent
research indicates that pyruvate also functions as an antioxidant,
inhibiting the production of harmful free radicals.
Certain microorganisms have been found to produce useful quantities
of pyruvate from glucose, an inexpensive substrate derived from
corn starch. The yeasts Debaryomyces coudertii (M. Moriguchi, Agr.
Biol. Chem. 46: 955-961 (1982)) and Saccharomyces exiguus (A.
Yokota et al., Agr. Biol. Chem. 48: 2663-2668 (1984)), for example,
are known to accumulate pyruvate, as are the basidiomycetes
Schizophyllum commune (S. Takao et al., J. Ferm. Tech. 60: 277-280
(1982)), and Agricus campestris (A. Yokota et al., Agr. Biol. Chem.
48: 2663-2668 (1984)). The yeast strain Torulopsis glabrata IFO
0005 was found to be a superior strain for the production of
pyruvate (T. Yonehara et al., J. Ferm. Bioeng. 78: 155-159 (1994)),
accumulating 67.8 g/L pyruvate in 63 hours (yield 0.494) in a
fed-batch fermentation with successive additions of glucose (R.
Miyata et al., J. Ferm. Bioeng. 82: 475-479 (1996)). A higher yield
(0.673) of pyruvate was observed in T. glabrata ACII-33, a mutant
with decreased pyruvate decarboxylase (PDC) activity (R. Miyata et
al., J. Biosci. Bioeng. 88: 173-177 (1999)). Decreased PDC activity
prevented the formation of acetate via acetaldehyde and thus
increased the pyruvate production. T. glabrata ACII-33 accumulated
60.3 g/L pyruvate in 47 hours in a 3 L jar fermenter.
Bacteria of the genera Corynebacterium (A. Yokota et al., Agr.
Biol. Chem. 48: 2663-2668 (1984)) and Acinetobacter (Y. Izumi et
al., Agr. Biol. Chem. 46: 2673-2679 (1982)), Enterobacter aerogenes
(A. Yokota et al., Agr. Biol. Chem. 48: 705-711 (1989)), and
Escherichia coli (A. Yokota et al., Appl. Microbiol. Biotech. 41:
638-643 (1994)) are also known to accumulate pyruvate. A lipoic
acid auxotroph of E. coli (strain W1485lip2) was found to produce
pyruvate aerobically from glucose under lipoic acid deficient
conditions. This strain accumulated 25.5 g/L pyruvate in 32 hours
with a yield of 0.51 in polypepton (4 g/L) supplemented media (A.
Yokota et al., Appl. Microbiol. Biotech. 41: 638-643 (1994)).
Alanine, which is derived from pyruvate, is an alpha amino acid
that is also commercially important, for example as a starting
material in the chemical industry. L-alanine is a chiral building
block being one of the smallest chiral compounds, with four
important functional groups: hydrogen, methyl, amino, and
carboxylic acid. Presently, alanine is produced using metabolically
engineered Corynebacterium or Brevibacterium bacterial strains in
which alanine dehydrogenase is overexpressed. However, the
efficiency of this method of production is limited because large
quantities of carbon move from pyruvate to acetyl-CoA and therefore
are unavailable for alanine production.
Diacetyl, also derived from pyruvate, is a flavoring/texture agent
for dairy products, and could find additional use in food
products.
These compounds (pyruvate, alanine and diacetyl) have current
market prices from $10 to $50/pound. Improved production methods
from renewable resources would open new markets for pyruvate and
its derivatives and thus reduce reliance on petroleum-derived
products.
SUMMARY OF THE INVENTION
The present invention is directed to a method for efficient
microbial production of pyruvate and its derivatives, such as
alanine and diacetyl. The method utilizes bacterial cells
exhibiting reduced activity of at least one enzyme in the pyruvate
dehydrogenase (PDH) complex of enzymes, compared to wild-type
bacterial cells. The bacterial cells are cultured in the presence
of a primary carbon source, preferably glucose, and a secondary
carbon source such as acetate and/or ethanol. If desired, the
bacterial cells can be cultured in the presence of an additional
carbon source, preferably a compound that is part of the
tricarboxylic acid cycle of the bacterial cell, such as
succinate.
Preferably PDH activity in the bacterial cells is undetectable. In
a particularly preferred embodiment of the invention, the method
utilizes bacterial cells wherein the gene encoding at least one
enzyme in the PDH complex of enzymes is knocked out.
When the method of the invention is employed to produce pyruvate,
pyruvate is preferably produced in an amount of at least about 30
g/L, and the yield of pyruvate as a function of glucose consumed is
preferably at least about 0.70.
Optionally the bacterial cells utilized in the method of the
invention exhibit reduced phosphoenolpyruvate carboxylase (PEP
carboxylase) activity or reduced pyruvate oxidase activity, or
both, compared to wild-type levels.
The production of pyruvate and diacetyl according to the invention
is not redox balanced and thus NADH will accumulate in the cells.
In these embodiments, the bacterial cells can be further engineered
exhibit increased or added NADH oxidase activity compared to
wild-type levels in order to maintain redox balance. When the
method of the invention is employed to produce diacetyl, the
bacterial cells also preferably exhibit added or increased
acetolactate synthase activity compared to wild-type cells.
The production of alanine according to the invention is redox
balanced and NADH will not usually accumulate in the cells.
Production of alanine can, however, be enhanced by utilizing
bacterial cells that exhibit added or increased alanine
dehydrogenase activity and/or reduced lactate dehydrogenase
activity compared to wild-type cells.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the biochemical pathways involved in the
accumulation of pyruvate, alanine and diacetyl. Not all biochemical
reactions or cofactors are shown.
FIG. 2 shows graphs of cell growth, metabolite production and feed
consumption as a function of time in fermentations using media C
for strains (a) CGSC4823; (b) CGSC4823 .DELTA.ppc; (c) CGSC6162;
and (d) CGSC6162 .DELTA.ppc.
FIG. 3 shows graphs of cell growth, metabolite production and feed
consumption as a function of time in fermentations using CGSC6162
at (a) pH of 6.0 and (b) pH of 7.0.
FIG. 4 shows a graph of fermentation as a function of time using
CGSC 6162 at 42.degree. C. and at a pH of 7.0.
FIG. 5 shows a graph of fermentation as a function of time using
CGSC 6162 .DELTA.ppc at 32.degree. C. and at a pH of 7.0.
FIG. 6 shows a graph of cell growth, alanine production and glucose
consumption as a function of time in a fermentation using CGSC6162
modified to overexpress the alaD gene from Bacillus sphaericus.
FIG. 7 shows a graph of cell growth, alanine production and glucose
consumption as a function of time in a fermentation using an ldhA
deletion mutant of CGSC6162 modified to overexpress the alaD gene
from B. sphaericus.
FIG. 8 shows a graph of the production of alanine in CGSC6162 ldhA
pTrc99A-alaD using modified growth parameters and supplemented with
additional NH.sub.4Cl: (a) glucose (O), alanine (.box-solid.),
pyruvate (.quadrature.); (b) OD (.circle-solid.), succinate
(.DELTA.), acetate (.gradient.).
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a process that utilizes bacterial cells for
the production of the metabolite pyruvate. The cells are grown,
preferably aerobically, in the presence of a primary carbon source
such as the carbohydrate glucose, a secondary carbon source such as
acetate or ethanol, and optionally a carbon source in the
tricarboxylic acid (TCA) cycle such as succinate.
The process can also be used to produce metabolites derived from
pyruvate (i.e., pyruvate derivatives), such as alanine and
diacetyl. Metabolites "derived from" pyruvate or "pyruvate
derivatives" are those biochemicals with respect to which pyruvate
is a metabolic precursor in bacterial metabolism. In other words,
the metabolic pathways for the production of biochemicals "derived
from" pyruvate branch away from the glycolytic/TCA pathway at
pyruvate. Examples of other products derived from pyruvate thus
include 2,3 butanediol, acetoin, isoleucine and valine. The general
microbial pathways for the synthesis of pyruvate and products
derived from pyruvate are shown in FIG. 1.
The PDH complex includes pyruvate dehydrogenase, dihydrolipoamide
acetyltransferase and dihydrolipoamide dehydrogenase. In E. coli, a
preferred microbe for use in the invention, these enzymes are
encoded by the aceE, aceF and lpd genes, respectively. The
bacterial cells used in the method of the invention exhibit reduced
activity of at least one enzyme in the pyruvate dehydrogenase (PDH)
complex of enzymes compared to wild-type cells. This is referred to
herein as reduced "PDH activity."
"Reduction" in an enzymatic activity compared to wild-type levels
of that activity in a cell includes, but is not limited to,
complete elimination of enzymatic activity. Thus, a reduction in
PDH activity compared to wild-type levels of PDH activity includes,
but is not limited to, complete elimination of PDH activity.
Complete "elimination" of PDH activity encompasses a reduction of
PDH activity to such an insignificant level that essentially no
carbon flows from pyruvate to acetyl CoA. Preferably, the bacterial
cells used in the method of the invention exhibit no detectable
activity of at least one enzyme in the PDH complex during the
entire period of fermentation. It should be understood that
although the method of the invention is not limited by the way in
which or the extent to which PDH activity is reduced in the
bacterial cells, it is preferred that PDH activity be completely
eliminated by disrupting the function of one or more genes
associated with PDH activity. In a preferred method of the
invention, bacterial cell growth surprisingly continues, with the
concomitant production of pyruvate and its derivatives, even though
PDH activity is completely eliminated.
Methods for reducing or eliminating PDH activity include those that
act directly on the gene encoding one or more of the PDH enzymes,
the mRNA transcript produced by the gene, the translation of the
mRNA transcript into the protein, or the abolishment of the
activity of the translated protein. One way the activity of an
enzyme can be reduced is by physically altering the gene encoding
the enzyme. For example, a gene encoding the enzyme can be mutated
using site-directed mutagenesis to introduce insertions, deletions
and/or substitutions. Alternatively, transcription of a gene can be
impeded by delivering to the cell an antisense DNA or RNA molecule
or a double stranded RNA molecule. Another way the activity of an
enzyme can be reduced is by interfering with the mRNA transcription
product of the gene. For example, a ribozyme (or a DNA vector
operably encoding a ribozyme) can be delivered to the cell to
cleave the target mRNA. Antisense nucleic acids and double stranded
RNAs may also be used to interfere with translation. Antibodies or
antibody-like molecules such as peptide aptamers can be used to
abolish the activity of the translated protein. In general, methods
that prevent production of an active PDH enzyme yield bacterial
cells that are referred to as "gene knockouts" as the term is used
herein.
Phosphoenolpyruvate carboxylase (PEP carboxylase) converts
phosphoenolpyruvate (PEP), the metabolic precursor to pyruvate, to
oxaloacetate and is encoded by the ppc gene. Accordingly, the
bacterial cells used in the method of the invention are optionally
further modified to reduce or eliminate the activity of PEP
carboxylase. The invention is not intended to be limited by the
method selected to reduce or eliminate PEP carboxylase
activity.
Pyruvate oxidase converts pyruvate into acetate and is encoded by
the poxB gene. Alternatively or in addition, the bacterial cells
are further modified to reduce or eliminate the activity of
pyruvate oxidase.
Hence, the method of the invention preferably utilizes bacterial
cells exhibiting, compared to wild-type cells, reduced or no PDH
activity (pdh.sup.-) and, optionally, one or more of reduced or no
pyruvate oxidase activity (pox.sup.-), reduced or no PEP
carboxylase activity (ppc.sup.-).
In the presence of only glucose as the carbon source, bacterial
cells deprived of PDH activity (so as to prevent conversion of
pyruvate to acetyl CoA) and, optionally, PEP carboxylase (so as to
prevent the precursor of pyruvate from being depleted) and,
optionally, pyruvate oxidase (so as to prevent conversion of
pyruvate to acetate) would certainly not be expected to grow, as
these modifications adversely affect the ability of the cells to
produce biochemical intermediates necessary for cell growth. In
particular, complete removal of PDH activity would prevent cells
from growing on glucose due to an inability to generate acetyl CoA,
.alpha.-ketoglutarate and succinate, all necessary for cell
growth.
The fact that the cells did grow when acetate was added as a
co-substrate was surprising as those skilled in the art of
bacterial fermentations view glucose as a preferred carbon source
over acetate. O'Beirne et al., Bioprocess Eng. 23:375-380 (2000),
discuss in detail the impact of acetate as a co-substrate in
continuous cultivations of E. coli, and conclude that acetate has a
noticeable inhibitory effect on the maximum specific growth rate
and CO.sub.2 evolution rate constant of E. coli even at low
concentrations. It is thus especially surprising to discover that
cells lacking PDH activity consume acetate, and grow well, when the
preferred substrate glucose is also available; i.e., that glucose
and acetate or ethanol will serve as simultaneous substrates.
Optionally the medium additionally contains a compound from the TCA
cycle such as succinate in order to fully meet the requirements of
the tricarboxylic acid (TCA) cycle.
In preferred embodiment, pyruvate production in the bacterial cells
according to the method exceeds at least about 20 g/L, preferably
about 30 g/L. Preferably the yield of pyruvate from glucose (grams
of pyruvate produced per gram of glucose consumed) of at least
about 0.70, more preferably at least about 0.75. Further, the
volumetric productivity of pyruvate according to the method is at
least about 1.0 g/liter-hour, more preferably at least about 1.5
g/liter-hour. Volumetric productivity is the pyruvate concentration
(in g/L) divided by the fermentation time required to attain that
concentration.
Regeneration of NAD is an important aspect in the aerobic growth of
E. coli and other microorganisms. Glycolysis is possible only if
NADH can be reoxidized since NAD is a necessary participant in the
oxidation of glyceraldehyde-3-phosphate. Typically, under aerobic
conditions, NADH is reoxidized by oxidative phosphorylation, a
membrane-bound process which generates ATP.
To our surprise, when E. coli cells lacking PDH activity were
cultured aerobically, lactate was formed. The formation of lactate
suggested that the enzyme lactate dehydrogenase (LDH), normally
observed in E. coli only under anaerobic conditions, may be active
in these aerobic cultures. The production of lactate in the aerobic
cultures further suggested that the cells are not able to oxidize
NADH fast enough even though they are grown aerobically. Because
the production of pyruvate and certain derivatives of pyruvate such
as diacetyl is not redox-balanced, the cells will accumulate NADH
during the operation of the method of the invention. As a result,
the pyruvate yield is expected to improve if reoxidation of NADH is
facilitated.
Overexpression of the enzyme NADH oxidase has been shown to result
in diminished flux through lactate dehydrogenase (LDH) because of
the removal of this enzyme's reduced cofactor NADH. Lopez DE Felipe
et al. (FEMS Microbiol. Lett. 156:15-19 (1998)) constructed an NADH
oxidase-overproducing Lactococcus lactis strain by cloning the
Streptococcus mutans nox-2 gene, which encodes the H.sub.2O-forming
NADH oxidase. This engineered system allowed a nisin-controlled 150
fold overproduction of NADH oxidase resulting in decreased NADH/NAD
ratio under aerobic conditions. In the presence of flavin adenine
dinucleotide (FAD), a cofactor required for NADH oxidase activity,
the lactate production was essentially abolished. Enhancing
reoxidation of NADH is thus expected to be accompanied by a
reduction in the formation of lactate, which is an undesirable
product.
Optionally, therefore, the bacterial cells used in the method of
the invention are further modified to increase the amount of NAD
regenerated or the rate at which NAD is regenerated. NADH can also
be directly oxidized using the nox gene which encodes NADH oxidase.
E. coli cells (or other cells that lack the nox gene) that have
been engineered to express additional NADH oxidase activity are
referred to herein as having "added" NADH oxidase activity. It is
expected that direct oxidation of NADH via NADH oxidase expression
will result in diminished or possibly abolished flux toward lactate
via LDH while ensuring the cells meet the demand of NAD. The
presence of NADH oxidase activity is also expected to reduce the
amount of lactate formed and therefore increase pyruvate yield. By
increasing the availability of NAD, the presence of NADH oxidase
activity is also expected to increase the rate of glucose
uptake.
Therefore, in embodiments of the method used to produce products
such as pyruvate or diacetyl which cause a net generation of NADH
due to a redox imbalance, the bacterial cells are further
optionally modified to increase the amount of, or rate at which,
NAD is regenerated from NADH. Preferably, a gene encoding NADH
oxidase (nox) is introduced into the bacterial cells. Such a gene
can be introduced on a plasmid or other vector, or can be
chromosomally integrated into the host genome.
A process that accumulates pyruvate is also expected to accumulate
biochemicals that are a few enzymatic steps from pyruvate. For
example, pyruvate is converted into alanine by a single enzyme,
alanine dehydrogenase. For alanine production, therefore, the
bacterial cells used in the method of the invention preferably
overexpress alanine dehydrogenase (alaD) in addition to exhibiting
reduced or eliminated PDH activity. As noted above, the bacterial
cells can be further modified to reduce or eliminate the activity
of pyruvate oxidase and/or PEP carboxylase.
The production of alanine is a redox-balanced synthesis in
bacterial cells, hence it is not recommended to add or increase
NADH oxidase activity. However, alanine production can also be
enhanced by reducing or eliminating the activity of lactate
dehydrogenase. Thus, the method for producing alanine optionally
utilizes bacterial cells exhibiting, compared to wild-type cells,
reduced or no lactate dehydrogenase activity (ldh.sup.-).
Diacetyl, another metabolite from pyruvate, can be produced by
expressing or overexpressing acetolactate synthase in bacterial
cells exhibiting reduced or eliminated PDH activity. Conversion of
pyruvate to acetolactate, which is catalyzed by acetolactate
synthase, is the first step in converting pyruvate to diacetyl. In
a preferred embodiment, the bacterial cells used to produce
diacetyl are E. coli cells that are preferably further modified to
exhibit added or increased acetolactate synthase activity and/or
added or increased NADH oxidase activity, and/or reduced or no
activity of pyruvate oxidase and/or PEP carboxylase, as described
above.
The present invention is illustrated by the following examples. It
is to be understood that the particular examples, materials,
amounts, and procedures are to be interpreted broadly in accordance
with the scope and spirit of the invention as set forth herein.
EXAMPLES
Example I
Accumulation of Pyruvate in Defined Minimal Media by E. coli
Mutants Lacking PDH Activity
Strains and plasmids. Strains and plasmids studied are listed in
Table 1. The strains fell into two groups. Members of the first
group were those that are blocked in their production of acetate.
These strains can be classified as rpoS mutants (AJW1483, CGSC5024,
CGSC6159), pta mutants (CGSC5992, CGSC7237), and ack mutants
(CGSC5993, CGSC7238) (CGSC: E. coli genetic stock culture. Yale
University). These strains were examined because it was
hypothesized that they might accumulate pyruvate if carbon were
prevented from entering the TCA cycle. Members of the second group
were those that possess mutations in genes in the PDH complex
(CGSC5518--a lpd mutant, CGSC4823--an aceE mutant, and CGSC6162--an
aceF mutant).
TABLE-US-00001 TABLE 1 Strains used Reference/ Name Genotype Source
MG1655 wild-type (F.sup.- .lamda..sup.-) Guyer et al., 1980 AJW1483
gal hft .DELTA. (rpoS::Kan) Contiero et al., 2000 CGSC4823 aceE2
tyrT58(AS) trp-26 mel-1 CGSC CGSC5024 F.sup.+ .lamda.-rpoS390(Am)
rph-1 CGSC CGSC5518 .lamda.-lpd-1 trpA58 trpE61 CGSC CGSC5992
.lamda.-pta-39 iclR7(const) trpR80 Guest, 1979 CGSC5993
.lamda.-gal- Guest, 1979 2trpA9761(Am)iclR7(const)trpR72(Am) ack-11
CGSC6159 .lamda.-rpoS396(Am) rph-1 CGSC CGSC6162 aceF10 fadR200
tyrT58(AS) adhE80 CGSC mel-1 CGSC7237 .lamda.-.DELTA. (his-gnd)861
hisJ701 pta-200 L.sub.EVine et al., 1980 CGSC7238 .lamda.-.DELTA.
(his-gnd)861hisJ701ackA200 L.sub.EVine et al., 1980 CGSC: E. coli
Genetic Stock Center, Yale University
Media and growth conditions. An initial comparison of strains
expressing endogenous PEP carboxylase was conducted using defined
minimal media modified from (Horn et al., Appl. Microbiol.
Biotechnol. 46:524-534 (1996)) containing (in units of g/L):
glucose, 30; KH.sub.2PO.sub.4, 6; (NH.sub.4).sub.2HPO.sub.4, 8;
citric acid, 0.3; MgSO.sub.4.7H.sub.20, 1.5; CaCl.sub.2.2H.sub.2O,
0.14; Fe.sub.2(SO.sub.4).sub.3, 0.0625; H.sub.3BO.sub.3, 0.0038;
MnCl.sub.2.4H.sub.2O, 0.0188; Na.sub.2EDTA.2H.sub.2O, 0.012;
CuCl.sub.2.2H.sub.2O, 0.0019; Na.sub.2MoO.sub.4.2H.sub.2O, 0.0031;
CoCl.sub.2.6H.sub.2O, 0.0031; Zn(CH.sub.3COO).sub.2.2H.sub.2O,
0.0099. Additionally, the media for CGSC 7237 and CGSC7238
contained 20 .mu.g/L histidine; for CGSC4823, CGSC5518 and CGSC5993
the media contained 20 .mu.g/L tryptophan; and for CGSC4823,
CGSC5518 and CGSC6162 the media contained 1 g/L acetate. All the
shake flasks were cultured at 37.degree. C. with 250 rpm
agitation.
Analytical methods. Cell growth was monitored by measuring the
optical density (OD) at 600 nm (DU-650 UV-Vis spectrophotometer,
Beckman Instruments), and this value was correlated to dry cell
mass. Samples were analyzed for glucose, pyruvate, acetate,
succinate and lactate quantitatively using a previous method (M.
Eiteman et al., Anal. Chim. Acia. 338: 69-75 (1997)).
Comparison of strains for growth and product formation. This
example employed a metabolic engineering approach for the
production of pyruvate. The strategy for generating pyruvate relied
on preventing this biochemical intermediate from entering the TCA
cycle or from being converted into acetate. We initially studied
eleven different strains of E. coli for acetate and pyruvate
accumulation and growth rate on glucose, looking for an absence of
acetate accumulation and/or relatively high pyruvate accumulation.
The initial specific growth rates were calculated from OD
measurements, and the concentrations of by-products found after
10-20 hours of growth were used to calculate product yields (Table
2).
The strains having a mutation all had growth rates lower than the
wild-type strain MG1655. In addition to the greatest growth rate,
MG1655 generated no pyruvate, and accumulated acetate to a yield of
0.11. The three rpoS strains each behaved differently with AJW1483
generating both pyruvate and acetate, CGSC5024 generating neither
pyruvate nor acetate, and CGSC6159 generating only acetate. Except
for CGSC7237, the pta or ack strains generated some acetate.
CGSC7237 and CGSC7238 accumulated significant pyruvate.
Of the strains with mutations in genes encoding for enzymes in the
pyruvate dehydrogenase complex, the lpd strain CSGC5518 was unable
to grow under the conditions studied. To our surprise, however, the
aceE and aceF strains CGSC4823 and CGSC6162 accumulated the
greatest concentrations of pyruvate, resulting in pyruvate yields
of 0.32 g/g and 0.40 g/g, respectively.
TABLE-US-00002 TABLE 2 Comparison of E. coli Strains for Growth and
Product Formation. Strain .mu.(h - 1) Y.sub.P/G (g/g) Y.sub.A/G
(g/g) MG1655 1.57 0.00 0.11 AJW1483 0.39 0.02 0.18 CGSC4823 0.91
0.32 * CGSC5024 1.17 0.00 0.00 CGSC5518 0.00 -- * CGSC5992 0.83
0.00 0.07 CGSC5993 0.75 0.00 0.17 CGSC6159 0.72 0.00 0.16 CGSC6162
0.45 0.40 * CGSC7237 0.51 0.08 0.00 CGSC7238 0.67 0.19 0.06 * media
contained acetate at an initial concentration of 1.0 g/L .mu. is
the initial specific growth rate, Y.sub.P/G is the mass pyruvate
yield on glucose and Y.sub.A/G is the mass acetate yield based on
glucose. Results are the means of triplicate experiments.
Example II
Accumulation of Pyruvate in Defined Minimal Media by E. coli
Mutants Lacking Both PDH and PEP Carboxylase Activity
PEP carboxylase is the enzyme that converts PEP to oxaloacetate.
This enzyme is believed not to be necessary for growth on acetate,
and we reasoned it would serve only to decrease the yield of
pyruvate by siphoning off its metabolic precursor PEP.
PEP carboxylase (ppc) mutants were generated from each of the five
strains that accumulated pyruvate or that did not generate acetate:
CGSC4823, CGSC5024, CGSC6162, CGSC7237, and CGSC7238. As noted in
Example 1, CSGC4823 is an aceE mutant (genotype: aceE2 tyrT58(AS)
trp-26 mel-1) and CGSC6162 is an aceF mutant (aceF10 fadR200
tyrT58(AS) adhE80 mel-1). As discussed in Example 1, CGSC6162
accumulates significant pyruvate even without additional genetic
modification and under nonoptimized conditions.
To construct these ppc mutants, a P1 lysate from JCL 1242
(.lamda.-F-.DELTA.(argF-lac)U169 ppc::Kan) was used to transduce
each strain to Kan(R) (Chao et al., Appl. Env. Microbiol.
59:4261-4265 (1993)). Because these ppc strains lacked the
anaplerotic enzyme PEP carboxylase, the media as described in
Example I was supplemented additionally with both acetate and
succinate. The acetate and succinate were supplied at initial
concentrations of either 0.62 g/L or 4.0 g/L each. The purpose of
this study was to determine the effect of ppc deletion on growth
rate and pyruvate formation, and the results are shown in Table
3.
TABLE-US-00003 TABLE 3 Comparison of E. coli ppc strains for growth
rate. Initial Succinate and Acetate .mu. Y.sub.P/G Q.sub.P Strain
(g/L) (h - 1) (g/g) (g/Lh) CGSC4823 .DELTA.ppc 0.62 0.03 0.14 0.03
4.0 0.04 0.08 0.03 CGSC5024 .DELTA.ppc 0.62 0.03 0.07 0.02 4.0 0.04
0.00 0.00 CGSC6162 .DELTA.ppc 0.62 0.04 0.76 0.28 4.0 0.13 0.72
0.15 CGSC7237 .DELTA.ppc 0.62 0.12 0.06 0.01 4.0 0.05 0.00 0.00
CGSC7238 .DELTA.ppc 0.62 0.11 0.11 0.03 4.0 0.05 0.00 0.00 .mu. is
the initial specific growth rate, Y.sub.P/G is the mass pyruvate
yield based on glucose and Q.sub.P is the volumetric productivity
of pyruvate.
Deletion of the ppc gene resulted in slower growth rates and
generally more pyruvate accumulation than the strains with ppc
gene. Except for CGSC6162 .DELTA.ppc, the maximum cell-mass
produced with 4.0 g/L initial concentrations was considerably
higher (.about.2 times) than with 0.62 g/L.
Strains CGSC5024 .DELTA.ppc, CGSC7237 .DELTA.ppc, and CGSC7238
.DELTA.ppc did not consume acetate when grown on 0.62 g/L initial
concentrations and in fact generated additional acetate once
succinate was consumed. These three strains also did not accumulate
pyruvate with 4.0 g/L initial concentrations and had the three
lowest pyruvate yields with 0.62 g/L initial concentrations.
CGSC4823 .DELTA.ppc and CGSC6162 .DELTA.ppc accumulated significant
pyruvate under both initial conditions. CGSC4823 .DELTA.ppc
produced less pyruvate than CGSC6162 .DELTA.ppc and also
accumulated acetate. CGSC6162 .DELTA.ppc did not accumulate
acetate. A small amount of lactate generation was also observed
with strains CGSC4823 .DELTA.ppc and CGSC6162 .DELTA.ppc. From this
study CGSC4823 .DELTA.ppc and CGSC6162 .DELTA.ppc were selected for
the fermenter studies.
Example III
Accumulation of Pyruvate in Rich Medium by E. coli Mutants Either
Lacking PDH Activity or Lacking Both PDH and PEP Carboxylase
Activity
Strains and plasmids. The strains studied were among those listed
in Table 1 in Example I. The strains included an rpoS mutant
(CGSC5024), pta mutant (CGSC7237), ack mutant (CGSC7238) and a
representative mutant for each of the three genes of the PDH
complex, aceE, aceF and lpd (CGSC4823, CGSC6162, CGSC5518,
respectively). A .DELTA.ppc mutation was introduced into each of
these strains as described in Example II.
Media and growth conditions. An initial comparison of strains was
conducted using 100 mL of media containing (g/L): glucose, 10.0;
acetic acid, 3.0; succinic acid, 6.0; yeast extract, 2.5; tryptone,
5.0; KH.sub.2PO.sub.4, 6.0; (NH.sub.4).sub.2HPO.sub.4, 8.0; citric
acid, 0.3; MgSO.sub.4.7H.sub.2O, 1.5; CaCl.sub.2.2H.sub.2O, 0.14;
Fe.sub.2(SO.sub.4).sub.3, 0.0625; H.sub.3BO.sub.3, 0.0038;
MnCl.sub.2.4H.sub.2O, 0.0188; Na.sub.2EDTA.2H.sub.2O, 0.012;
CuCl.sub.2.2H.sub.2O, 0.0019; Na.sub.2MoO.sub.4.2H.sub.2O, 0.0031;
CoCl.sub.2.6H.sub.2O, 0.0031; Zn(CH.sub.3COO).sub.2.2H.sub.2O,
0.0099. All the 500 mL shake flasks were cultured in duplicate at
37.degree. C. with 250 rpm agitation and initial pH of 7.0.
Subsequent studies of selected strains were conducted in
computer-controlled fermentations of 1.5 L volume carried out in
2.5 L fermenters (Bioflow III, New Brunswick Scientific Co.,
Edison, N.J.). Unless otherwise stated, the temperature was
maintained at 37.degree. C., agitation at 750 rpm, sterile filtered
air was sparged at a rate of 1.5 L/min, and 20% NaOH and 20% HCl
were used to control pH. The level of aeration ensured that the
dissolved oxygen never fell below 30% of saturation for any of the
fermentations. Samples were taken periodically and stored at
-20.degree. C. for subsequent analysis. The medium was identical to
that described above except for initial concentration of 40 g/L
glucose. For fed batch fermentations, glucose concentration was
maintained at 3 g/L by automatic feeding of a 600 g/L glucose
solution (YSI, Yellow Springs, Ohio).
Comparison of strains for growth and product formation. This
example employed a metabolic engineering approach for the
production of pyruvate. The strategy for generating pyruvate relied
on preventing this biochemical intermediate from entering the TCA
cycle or from being converted into acetate. Therefore, we initially
studied six strains of E. coli and their corresponding ppc mutants
for growth and acetate and pyruvate accumulation using a medium
containing 10 g/L glucose. For all these strains, the glucose was
exhausted in 7-10 hours. Table 4 summarizes the results of these
duplicate shake flask fermentations, with pyruvate and acetate
yields calculated at the time that glucose was exhausted.
TABLE-US-00004 TABLE 4 Pyruvate and acetate mass yields in strains
of Escherichia coli. Strain Max. OD Y.sub.P/G Y.sub.A/G CGSC4823
8.1 0.54 -0.01* CGSC4823 .DELTA.ppc 5.1 0.097 0.19 CGSC5024 8.8
0.085 0.34 CGSC5024 .DELTA.ppc 4.7 0.12 0.28 CGSC5518 6.1 0.31 0.11
CGSC5518 .DELTA.ppc 8.0 0.083 0.30 CGSC6162 6.8 0.47 0.030 CGSC6162
.DELTA.ppc 6.9 0.41 0.029 CGSC7237 6.7 0.34 0.024 CGSC7237
.DELTA.ppc 5.5 0.11 0.30 CGSC7238 7.0 0.25 0.17 CGSC7238 .DELTA.ppc
5.3 0.10 0.23 Y.sub.P/G: pyruvate generated/glucose consumed (g/g)
Y.sub.A/G: acetate generated/glucose consumed (g/g) *For this
growth condition and time interval, the organism consumed
acetate
Strains with alterations in acetate synthesis included CGSC5024
(rpoS), CGSC7237 (pta) and CGSC7238 (ack). Of these CGSC7237 and
CGSC7238 accumulated greater than 25% (mass yield) pyruvate, but
only CGSC7237 generated less than 15% acetate. Introduction of the
ppc mutation increased pyruvate accumulation only with CGSC5024,
the lowest pyruvate producer. With CGSC7237 a ppc mutation
increased acetate yield twelve-fold and decreased pyruvate yield
three-fold. For all three strains a deletion in ppc substantially
reduced the maximum cell concentration and hence cell yield.
Strains with mutations in the PDH complex included CGSC4823 (aceE),
CGSC6162 (aceF) and CGSC5518 (lpd). Each of these strains initially
consumed acetate, accumulated significant pyruvate and began
accumulating acetate after 2-4 hours. By the time glucose was
exhausted, only CGSC4823 had a small net consumption of acetate.
Introduction of the ppc mutation decreased pyruvate yield for all
three strains, and increased acetate yield in the CGSC4823 and
CGSC5518. A deletion in ppc increased cell yield for CGSC5518,
decreased the cell yield for CGSC4823, and had no effect on cell
yield in CGSC6162.
Example IV
Batch Fermentation Studies on CGSC4823, CGSC4823 .DELTA.ppc,
CGSC6162 and CGSC6162 .DELTA.ppc
CGSC4823 .DELTA.ppc and CGSC6162 .DELTA.ppc, along with their
parent strain (CGSC4823 and CGSC6162) were grown at 1.5 L (initial
volume) in a 2.5 L fermenter (New Brunswick Scientific Instruments,
NJ) at 37.degree. C. and 750 rpm with 1.5 L/min constant air
flowrate using three different media (Media A, Media B and Media
C). The modified Horn medium (Example I) was again used but with
different initial concentrations of various carbon sources. Medium
A contained 20 g/L glucose, 1 g/L acetate and 2 g/L succinate.
Medium B contained 40 g/L glucose, 3 g/L acetate and 6 g/L
succinate. Medium C contained 40 g/L glucose, 3 g/L acetate, 6 g/L
succinate, 2.5 g/L yeast extract and 5 g/L tryptone. When the
glucose concentration decreased to 3.0 g/L, it was controlled at
this concentration by the automatic feeding of a 600 g/L glucose
solution using an on-line glucose analyzer (YSI Instruments, OH).
Samples were taken periodically during growth and stored at
-20.degree. C. for subsequent analysis. Analytical methods were as
in Example I.
The results are shown in Table 5. Cells generally grew to their
lowest cell mass in media A because of the lower concentrations of
acetate and succinate in the media. Supplementing the media with
yeast extract and tryptone (Media C) reduced the lag phases and
generally increased the growth rates. FIG. 2 shows the products of
fermentations using Media C and the strains (a) CGSC4823, (b)
CGSC4823 .DELTA.ppc, (c) CGSC6162 and (d) CGSC6162 .DELTA.ppc,
respectively. In these fermentations the pyruvate yield was greater
than 0.50 except for CGSC4823 .DELTA.ppc. Very small amounts of
lactate were observed in CGSC4823 .DELTA.ppc and CGSC6162
.DELTA.ppc. From this study, CGSC6162 and CGSC6162 .DELTA.ppc were
selected for further fed-batch fermentation studies using Media
C.
TABLE-US-00005 TABLE 5 Comparison of E. coli strains for growth
rate and product formation when grown on different media. Media
.mu. Max. Pyr. Y.sub.P/G Strain (g/Lh) (h.sup.-1) Max. OD (g/L)
(g/g) Q.sub.P CGSC4823 A 0.18 4.1 9.8 0.58 0.28 B 0.10 5.0 17.0
0.46 0.29 C 0.20 11.0 20.0 0.52 1.10 CGSC4823 .DELTA.ppc A 0.08 2.2
10.0 0.63 0.10 B 0.04 7.5 16.0 0.41 0.29 C 0.07 22.3 1.1 0.03 0.06
CGSC6162 A 0.17 7.2 11.0 0.56 0.69 B 0.11 13.0 18.0 0.53 0.56 C
0.11 10.0 23.0 0.63 1.50 CGSC6162 .DELTA.ppc A 0.03 0.7 3.7 0.91
0.09 B 0.02 0.4 0.4 -- -- C 0.19 11.0 24.0 0.64 1.10 Media A: 20
g/L glucose, 1 g/L acetate, 2 g/L succinate Media B: 40 g/L
glucose, 3 g/L acetate, 6 g/L succinate Media C: 40 g/L glucose, 3
g/L acetate, 6 g/L succinate, 2.5 g/L yeast extract, 5 g/L tryptone
.mu. is the initial specific growth rate, Y.sub.P/G is the mass
pyruvate yield based on glucose and Q.sub.P is the volumetric
productivity of pyruvate.
Example V
Fed-Batch Fermentation Studies on CGSC6162 and CGSC6162.DELTA.ppc
to Study the Effect of pH
We next conducted fed-batch fermentations with CGSC6162 and
CGSC6162 .DELTA.ppc to study the affect of pH on pyruvate
accumulation. The fermentations again commenced at a pH of 7.0 with
a glucose concentration of 40 g/L. After 12 hours of growth, the pH
was shifted (over the course of about 30 minutes) to the desired
constant pH. When the glucose concentration reached 3.0 g/L (at
16-18 hours), glucose was maintained at that concentration until
the fermentations terminated at 36 hours. Table 6 shows the mean
specific rates of glucose consumption and formation for the three
products at the pH levels studied over the time interval of 12
hours to 36 hours. The results include the specific activities of
LDH and POX at 20 hours.
The pH had a significant effect on the CGSC6162 fermentations. At
the lowest pH (6.0), glucose consumption was low, and acetate was
the exclusive product (with a mass yield of 67%, essentially the
theoretical maximum). Also, the activity of POX was relatively
high. At the other three levels of pH (6.5, 7.0, 7.5), additional
acetate did not form, pyruvate was the primary product, but lactate
formation was also significant. The activity of POX was 3-4 times
lower than observed at a pH of 6.0. For all CGSC6162 fermentations
during the initial phase (pH 7.0 until 12 hours), pyruvate mass
yield was approximately 70%. Thereafter the pyruvate yield
decreased with time. For example, during the interval 12-20 hours,
the yields were 59% (pH of 6.5), 72% (7.0), 52% (7.5), while during
the interval 20-28 hours the yields were 25% (6.5), 32% (7.0), 38%
(7.5). In the pH range of 6.5-7.5, lactate accumulated only after
the acetate was exhausted. The maximum pyruvate concentration
achieved was about 35 g/L for the fermentations continuously at a
pH of 7.0.
For the CGSC6162 .DELTA.ppc fermentations at the lowest pH of 6.0,
the pyruvate that had been formed during the first 12 hours at a pH
of 7.0 was partly consumed. Acetate was the exclusive product, and
the POX activity was high. At pH values of 6.5 and 7.0, acetate was
still formed, but pyruvate was the principal product and POX
activity was about half that observed at a pH of 6.0. At a pH of
6.5 and 7.0, the POX activity was about two times greater in
CGSC6162 .DELTA.ppc than in CGSC6162. For the CGSC6162 .DELTA.ppc
fermentations during the initial phase (pH 7.0 until 12 hours),
pyruvate mass yield was about 75% and again, the pyruvate yield
decreased thereafter during the course of the fermentations. The
LDH activity was low in all cases, did not correlate with lactate
formation, and did not appear to follow any trend with pH.
TABLE-US-00006 TABLE 6 Specific rates of glucose consumption and
pyruvate, acetate and lactate generation during fed-batch
fermentations of CGSC6162 and CGSC6162 .DELTA.ppc at different
levels of pH. First 12.0 hours of growth occurred at a pH of 7.0,
after which time the pH was gradually changed to indicated pH for
an additional 24 hours and the rates recorded. Enzyme activities at
20 hours are in U/mg protein. Strain pH q.sub.G q.sub.P q.sub.A
q.sub.L LDH POX CGSC6162 6.0 0.34 0 0.23 0 0.08 2.25 6.5 0.94 0.27
0 0.26 0.08 0.51 7.0 0.89 0.34 0 0.18 0.06 0.51 7.5 0.80 0.31 0
0.21 0.09 0.77 CGSC6162 .DELTA.ppc 6.0 0.32 -0.15* 0.30 0 0.08 1.92
6.5 0.57 0.21 0.13 0 0.01 1.04 7.0 0.60 0.32 0.06 0.05 0.00 1.07 q:
specific rate of formation/consumption during the time interval of
12 hours to 36 hours (g compound/g cells hour) *For this growth
condition and time interval, the organism consumed this
compound
Several remarkable results are shown in FIG. 3. E. coli CGSC6162
did indeed simultaneously consume acetate and glucose.
Interestingly, during the initial portion of these fermentations,
these strains consumed all the acetate supplied, and the maximum
concentrations of pyruvate occurred when the acetate concentration
reached zero. Cell growth ceased when the acetate became depleted 8
hours after inoculation, demonstrating that this substrate was
necessary for cell growth. Even though acetate was depleted in less
than 10 hours, the cells generated well over 30 g/L pyruvate, and
the mass yield of pyruvate from glucose was about 0.70 during the
first 20 hours. The volumetric productivity during this time
interval of low cell density was over 1.5 g/L hour.
After 20 hours, lactate surprisingly appeared as a co-product with
acetate, with the concentration of pyruvate diminishing. Lactate
(and NAD) generation from pyruvate (and NADH) by lactate
dehydrogenase is known to be used by E. coli as a means to balance
the cofactors NADH and NAD (Gokarn et al., Appl. Env. Microbiol.
66:1844-1850 (2000)). It therefore is particularly interesting that
lactate was synthesized during this aerobic fermentation in which
NADH could generate energy for the cell, as it suggests that NAD
could not be regenerated from NADH quickly enough via oxidative
phosphorylation to meet the demand of glucose uptake through the
EMP pathway.
An interesting observation is that the generation of carbon dioxide
was much lower than commonly observed in aerobic fermentations,
which can be explained as follows. During the production of
pyruvate which appears to have come from 70% of the glucose in our
study, no carbon dioxide is generated from the conversion of
glucose to pyruvate. One mole of carbon dioxide is generated from
each mole of carbon entering the pentose phosphate pathway (likely
less than 15% of the total glucose), and only a small quantity of
carbon dioxide will be generated from the consumption of acetate
through the TCA cycle, primarily for toward the synthesis of
biomass.
The results demonstrate that a pyruvate yield exceeding 0.75 was
routinely obtained during the first 12 hours of these
fermentations. Moreover, the results demonstrate that changing the
pH from 7.0 after 12 hours did not improve the pyruvate yield.
Switching to a pH of 6.0 for both strains resulted in the
consumption of pyruvate and generation of acetate. This utilization
of pyruvate did not result in an increase in the cell mass
concentration. FIG. 3(a) shows the fermentation using CGSC6162 at a
pH of 6.0. FIG. 3(b) shows the fermentation using CGSC6162 at a pH
of 7.0. In cases in which the pH remained at 7.0, the volumetric
productivity of pyruvate at the time acetate was depleted was over
1.5 g/Lh. This is the first report of a viable industrial process
for the production of pyruvate in E. coli, something that was
heretofore thought impossible because of the complexities and
interdependencies of the interrelated metabolic pathways that stem
from the pyruvate node.
Example VI
Fed-Batch Fermentation Studies on CGSC6162 and CGSC6162.DELTA.ppc
to Study the Effect of Temperature
Fed-batch fermentations of CGSC6162 and CGSC6162 .DELTA.ppc were
also studied at three different temperatures (32.degree. C.,
37.degree. C. and 42.degree. C.) at pH 7.0 for 36 hours. Pyruvate
and lactate yields were calculated for two time intervals (0-20
hours; and 20-36 hours), and the results are shown in Table 7.
CGSC6162 did not accumulate acetate at any temperature (FIG. 4
shows a 42.degree. C. fermentation). Lactate and pyruvate
production were strongly influenced by temperature. The initial
rate of pyruvate formation was greatest at 42.degree. C., with
21-25 g/L accumulating in 12 hours. However, after about 12 hours
at this temperature, the cell density decreased, and CGSC6162
accumulated lactate instead of pyruvate. Thus, the greatest
pyruvate concentrations and lowest lactate concentrations over the
course of 36 hours were achieved when the fermentation temperature
was maintained at 32.degree. C.
Although initially CGSC6162 .DELTA.ppc consumed acetate, this
strain eventually accumulated acetate at all temperatures (FIG. 5
shows an example CGSC6162 .DELTA.ppc fermentation at 32.degree.
C.). However, the accumulation of acetate was greater and commenced
sooner at higher temperature. Specifically, at 42.degree. C.
acetate began accumulating at about 8 hours, at 37.degree. C.
acetate accumulation began at 12-16 hours, and at 32.degree. C.
acetate began accumulating after 20 hours. Similar to CGSC6162,
with CGSC6162 .DELTA.ppc lactate accumulation began after 12 hours
for all temperatures. The rate of lactate production was again
strongly temperature dependent, with higher temperature favoring
lactate. This strain also achieved its maximum pyruvate
concentration at 32.degree. C.
Table 7 shows the specific activities of LDH and POX at 20 hours
for the two strains at the three different temperatures. In all
cases the LDH activity was low and did not follow a trend. POX
activity tended to increase with increasing temperature in both
CGSC6162 and CGSC6162 .DELTA.ppc. Furthermore, POX activity was
about twice as great in CGSC6162 .DELTA.ppc than in CGSC6162 at
37.degree. C. and 42.degree. C. As the data indicates, the
production of pyruvate may further be increased by deleting the pox
gene.
TABLE-US-00007 TABLE 7 Yields of pyruvate and lactate during
fed-batch fermentations of CGSC6162 and CGSC6162 .DELTA.ppc at
different controlled temperatures. Yields are calculated during two
time intervals (0-20 hours and 20-36 hours). Enzyme activities at
20 hours are in U/mg protein. Temp Maximum Pyruvate Y.sub.P/G
Y.sub.L/G Strain (.degree. C.) Concentration (g/L) 0-20 h 20-36 h
0-20 h 20-36 h LDH POX CGSC6162 32 37 0.73 0.47 0.03 0.07 0.04 0.57
37 36 0.67 0.25 0.08 0.20 0.06 0.51 42 32 0.67 0.16 0.01 0.58 0.07
0.80 CGSC6162 .DELTA.ppc 32 35 0.70 0.60 0.01 0.07 0.01 0.45 37 35
0.74 0.41 0.02 0.11 0.00 1.07 42 29 0.57 0.25 0.06 0.60 0.01 1.58
Y.sub.P/G: pyruvate generated/glucose consumed (g/g) Y.sub.L/G:
lactate generate/glucose consumed (g/g)
Example VII
Overexpression of NADH Oxidase to Produce Pyruvate
The accumulation of small amounts of lactate during highly aerobic
conditions (see Example V and VI) suggests that NADH is not being
converted into NAD to keep pace with the demand of glycolysis. It
would be of little benefit to delete lactate dehydrogenase activity
as a means of producing pyruvate and avoiding lactate generation,
as this does not address what appears to be the underlying cause,
namely, the conversion of NADH to NAD. Furthermore, cells do not
appear to be limited in ATP generation, since the NADH is being
consumed toward lactate formation rather than via oxidative
phosphorylation.
One way to enhance the regeneration of NAD is by introducing
additional NADH oxidase activity into the strains. The nox gene
encodes NADH oxidase which converts NADH and oxygen directly into
NAD and water (without the generation of ATP). Known nox genes
encoding for NADH oxidase include those from Streptococcus
pneumoniae, S. faecalis and Saccharomyces cerevisiae. Lopez de
Felipe et al. (FEMS Microbiol. Lett. 156:15-19 (1998)) have
previously used NADH oxidase overexpression in Lactococcus lactis
to significantly decrease the NADH/NAD ratio and reduce lactate
synthesis. We have constructed a pTrc99A-nox plasmid which
overproduces NADH oxidase from S. pneumoniae. This plasmid, which
is inducible by the addition of IPTG, can be used to transform PDH
and PDH+PEP carboxylase mutant strains to enhance the production of
pyruvate.
Example VIII
Enhanced Production of Diacetyl
If pyruvate can accumulate significantly in cells, then biochemical
derivatives of pyruvate might also accumulate in these cells.
Diacetyl(2,3-butanedione), with a vapor pressure similar to
ethanol, is a constituent of food and fruit aromas and is the main
constituent of "butter aroma." As shown in FIG. 1, the synthesis of
diacetyl first involves the conversion of pyruvate to acetolactate
by the enzyme acetolactate synthase. Diacetyl can be synthesized
from pyruvate by a two step process involving 1) the conversion of
pyruvate to acetolactate by the enzyme acetolactate synthase and 2)
the chemical oxidation/decomposition of acetolactate to diacetyl.
These two additional pathways are shown in FIG. 1.
Plasmid pAAA215 overproduces acetolactate synthase from Bacillus
subtilis (Aristidou et al., Biotechnol. Bioeng. 44:944-951 (1994)).
The synthesis of this enzyme appears to be induced by cell growth
and its activity is stimulated by the presence of acetate
(Holtzclaw et al., J. Bacteriol. 121:917-922 (1975)). The product
acetolactate itself is chemically unstable, being oxidized (by
oxygen) to diacetyl in the presence of metal ions such as
Fe.sup.3+. Two competing biochemical reactions can also occur. In
some organisms, acetolactate decarboxylase catalyzes the
decarboxylation of acetolactate under oxygen limited conditions to
acetoin. The chemical oxidation of acetolactate appears to be
favored at a pH of about 5, while the enzymatic decarboxylation of
acetolactate appears to be favored at a pH of about 6.5. The
presence of acetolactate decarboxylase activity in E. coli has not
been established. A second competing reaction involves the enzyme
diacetyl reductase, whose activity has been observed in E. coli,
which directly converts diacetyl to acetoin. This reaction has a pH
optimum of about 7.0, and requires NADH or NADPH, the former being
more active with the latter. Interestingly, acetate has been shown
to inhibit the activity of this enzyme, 20 mM decreasing the
activity by 35% (Ui, Agr. Biolog. Chem. 51:1447-1448 (1987)).
Moreover, under highly oxygenated conditions, NADH and NADPH will
normally be less prevalent than their oxidized analogues. The pH
optima, the effects of acetate and oxygen, and the prospects for
adding other chemical catalysts or inhibitors would suggest that a
fermentation process to accumulate diacetyl is feasible under the
general conditions we have previously observed for pyruvate
generation.
To accomplish, this, the "best" strains as identified in Example I
are transformed with the pAAA215 plasmid that overproduces
acetolactate synthase from B. subtilis (Aristidou et al.,
Biolechnol. Bioeng. 44:944-951 (1994)). The plasmid pTrc99A-nox,
which overproduces NADH oxidase, can also be transformed into these
strains. Because they contain compatible replicons, both the
pTrc99A-nox plasmid which overproduces NADH oxidase and the pAAA215
plasmid which overproduces acetolactate synthase can be introduced
into the same cell. The pTrc99A-nox plasmid uses the colE1 replicon
and is selected for using ampicillin while the pAAA215 plasmid uses
the P15A replicon and is selected for using tetracycline. Numerous
researchers have constructed dual plasmid strains like this where
the first plasmid contained the colE1 replicon and the second
plasmid contained the P15A replicon. The result is the construction
of at least one strain with a PDH mutation and enhanced
acetolactate synthase activity, and also a strain with additional
increased NADH oxide activity.
The chemical production of diacetyl is promoted by oxidation. The
conversion of diacetyl to acetoin by the undesirable enzymatic
reaction (diacetyl reductase) uses NADPH or NADH as a cofactor with
the former being preferred. We would expect a highly oxygenated
environment to prevent this undesirable reaction. It does not
appear feasible to perform a gene "knock out" of diacetyl reductase
as evidence suggests that this reaction is carried out
inadvertently (i.e., nonspecifically) by one or more general
reductase enzymes. The presence of NADH oxidase should facilitate
diacetyl production because, like pyruvate generation itself, a
greater rate of NADH oxidation would tend to increase the rate of
glucose uptake and reduce the availability of NADH/NADPH for side
reactions.
Example IX
Enhanced Production of Alanine
Alanine can be synthesized from pyruvate in a single reaction step
by the enzyme alanine dehydrogenase. L-alanine is generally
produced by an enzymatic process in which L-aspartic acid is
enzymatically decarboxylated (Ichiro et al., U.S. Pat. No.
3,458,400 (1969)). E. coli has activity in a racemase, which would
convert the L-alanine produced in any process to D-alanine,
resulting in a DL-alanine product. Although ultimately the activity
of alanine racemase can be abolished to yield exclusively the
L-alanine product, this example focuses on the production of the
racemic mixture of alanine.
In order to test whether the same approach could be used to
generate derivatives of pyruvate, such as alanine, we transformed
CGSC6162 with the pTrc99A-alaD plasmid that we constructed which
overproduces alanine dehydrogenase from Bacillus sphaericus
(Ohashima et al., Eur. J. Biochem. 100:29-39 (1979)). Alanine
dehydrogenase is an enzyme that converts pyruvate to alanine. This
strain was grown on media containing 25 g/L glucose, 3.0 g/L yeast
extract, 6.0 g/L tryptone, 2.7 g/L succinate, 1 g/L acetate, 1 mg/L
biotin, 1 mg/L thiamine HCl, 15 mg/L CaCl.sub.22H.sub.2O, 5.875 g/L
Na.sub.2HPO.sub.4, 3.0 g/L KH.sub.2PO.sub.4, 6 g/L NH.sub.4Cl and
0.25 g/L MgSO.sub.47H.sub.2O. Continuous constant agitation (1000
rpm) and air flowrate (1.0 L/min) were used.
FIG. 6 shows the results, with alanine accumulating to 3.3 g/L.
Alanine accumulated to this level even though the nitrogen
concentration (as N) was less than 2 g/L in the initial media. This
nitrogen would be required both for cell growth (about 12% N) and
alanine synthesis (about 15% N). The activity of alanine
dehydrogenase was 0.04 U/mg at 8 hours and 0.06 U/mg at 14
hours.
The formation of alanine consumes NADH in the final step via
alanine dehydrogenase. Considering that the cell would otherwise
generate ATP from the oxidative phosphorylation of NADH, and full
aeration was used in our study with dissolved oxygen concentration
always above 75% saturation, this level of accumulation is
remarkable.
Interestingly, significant pyruvate still accumulated (10 g/L).
This suggests that alanine production was limited due to a
non-optimal amount of alanine dehydrogenase. The activity of
alanine dehydrogenase in our system can be increased by recloning
the B. sphaericus alaD gene into a pTrc99A derivative that we have
constructed which expresses genes at a level 3-10 times higher than
is obtainable in the original pTrc99A vector.
The biochemistry of alanine production has a crucial difference
from the biochemistry of pyruvate production. The production of
pyruvate from glucose generates 2 moles of NADH per mole of
glucose, and should be conducted with high oxygenation so the NADH
produced can be converted to ATP by oxidative phosphorylation.
Moreover, oxygen limitation in this case could lead to activation
of pyruvate formate lyase and subsequent reduction of pyruvate
through fermentative regeneration of NAD. In contrast, the overall
production of alanine from glucose does not generate NADH due to
the final step from pyruvate to alanine. Since oxygenation affects
the NADH/NAD balance, the availability of oxygen should be have a
significant impact on alanine production. We expect that reduction
of oxygen availability should improve alanine production until a
level is reached where the fermentative enzymes such as lactate
dehydrogenase and pyruvate formate lyase are induced. The oxidation
"state" of the system can be most readily monitored by dissolved
oxygen concentration or by the culture's redox potential.
Note that because alanine dehydrogenase consumes NADH, NADH
production is balanced during the conversion of glucose to alanine.
It would therefore be unnecessary, and indeed undesirable, to
overexpress NADH oxidase to enhance the production of the pyruvate
derivative alanine.
Example X
Enhanced Production of Alanine in a Lactate Dehydrogenase
Mutant
Lactate dehydrogenase converts pyruvate to lactate and thus
competes with alanine dehydrogenase, which converts pyruvate to
alanine. The lactate dehydrogenase and alanine dehydrogenase
enzymes both use pyruvate and NADH as substrates; therefore, if
native lactate dehydrogenase is present during a fermentation in
which alanine is the desired product, the lactate dehydrogenase
could undesirably compete with alanine dehydrogenase.
In order to prevent lactate dehydrogenase from possibly competing
with alanine dehydrogenase in the generation of the pyruvate
derivative alanine, we constructed an ldhA deletion mutant of
CGSC6162. In E. coli, ldhA is the gene that encodes lactate
dehydrogenase. We also further improved the alanine production
process regarding oxygenation by curtailing agitation during the
course of the fermentation as suggested in Example IX. The
ldhA::Kan deletion mutant from the E. coli strain NZN111 (Bunch et
al., Microbiology, 143:187-195 (1997)) was introduced into CGSC6162
by P1 phage transduction. We transformed this CGSC6162 ldhA
deletion mutant with the pTrc99A-alaD plasmid that we constructed
which overproduces alanine dehydrogenase from Bacillus
sphaericus.
CGSC6162 ldhA::Kan pTrc99A-alaD cells were grown in a BioFlow 2000
fermenter (New Brunswick Scientific Co., New Brunswick, M.J.), with
1.5 L of media containing 40.0 g/L glucose, 6.0 g/L succinic acid,
3.0 g/L acetic acid, 10 g/L tryptone, 2.5 g/L yeast extract, 3.0
g/L KH.sub.2PO.sub.4, 6.0 g/L NaH.sub.2PO.sub.4, 6.0 NH.sub.4Cl,
0.14 g/L CaCl.sub.2.2H.sub.2O and 0.25 g/L MgSO.sub.4.7H.sub.2O,
and 100 mg/L ampicillin. The fermenter was operated at 37.degree.
C., a pH of 7.0, with 1000 rpm agitation and 1.0 L/min air flow.
After 4.0 hours of growth in the fermenter, IPTG was added to a
final concentration of 1.0 mM. At 11.0 hours of growth in the
fermenter, the agitation was reduced to 250 rpm. At 15.0 hours of
growth in the fermenter, an additional 110 mL volume of solution
was added to the fermenter containing 30 g glucose and 7.5 g
NH.sub.4Cl.
FIG. 7 shows the results, with alanine accumulating to 12 g/L.
These results show that the deletion of ldhA in CGSC6162 does not
deleteriously impact cell growth. Furthermore, these results
demonstrate that additional improvement in the production of
pyruvate derivatives such as alanine can be attained both by
genetic means (ldhA mutation) and by process modifications (optimal
oxygenation).
Increased Yield with Increased Ammonium
By altering growth conditions and supplying additional ammonium 10
chloride, the yield of alanine increased to 32 g/liter. See M. Lee
et al., Appl. Microbiol. Biotechnology 65: 56-60 (2004).
Specifically, cells of CGSC6162 ldhA pTrc99A-alaD were first grown
at 20 mL volume in an agitated screw top test tube with media
composed of (per liter) 15.0 g glucose, 3.0 g acetic acid, 6.0 g
succinic acid, 2.5 g tryptone, 2.5 g NaCl, and 1.25 g yeast
extract. After 3 hours of growth 10 mL was used to inoculate 100 mL
of media in a 250 mL baffled shake flask composed of (per liter)
15.0 g glucose, 3.0 g acetic acid, 6.0 g succinic acid, 10.0 g
tryptone, 2.5 g yeast extract, 3.0 g KH.sub.2PO4, 6.0 g
Na.sub.2HPO4, 6.0 g NH.sub.4Cl, 0.14 g CaCl.sub.2.H.sub.2O, and
0.25 g MgSO.sub.4.7H.sub.2O. Cells were grown at 250 rpm (19 mm
radius of orbit) for 6 hours and then used to inoculate a fermenter
of the same composition as the shake flask except 40 g/liter
glucose. Fermentations of 1.5 liter initial volume were conducted
using a BioFlow 2000 (New Brunswick Scientific Company, New
Brunswick, N.J.). Air was supplied continuously at 1.0
liter/minute. After 3-4 hours of growth in the fermenter, 1.0 mM
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) was added for gene
induction. During the first 11 hours of fermentation, the agitation
was 1000 rpm, a rate which insured that the dissolved oxygen
remained above 20% of saturation. At 11 hours the alanine
production phase was initiated by reducing the agitation rate to a
lower constant value as described in the text. Oxygen mass transfer
coefficients (k.sub.La) for each experimental agitation rate were
determined in a separate experiment using the static sparging
method (W. S. Wise, J. Gen. Microbiol. 5: 167-177 (1951)) with
identical media and fermenter system. At 15 hours, additional
glucose and NH.sub.4Cl was added as described in the text to
replenish these components that had been consumed for the
generation of cell mass and alanine. All media contained 100
mg/liter ampicillin and were carried out at 37.degree. C. and a pH
of 7.0 controlled throughout the fermentations.
In studies on the effect of k.sub.La on alanine accumulation, a
consistent result was that alanine generation occurred at the
highest rate immediately following the addition of glucose and
NH.sub.4Cl at 15 hours. In order to determine whether NH.sub.4Cl
was limiting the conversion of pyruvate to alanine via alanine
dehydrogenase, we determined the ammonium ion concentration at the
end of these fermentations, and found the ammonium concentration to
be a minimum of 20 mmol/liter. We then repeated those fermentations
with the lowest value of k.sub.La of 7 hour.sup.-1. In this case,
however, we provided three times the NH.sub.4Cl (22.5 g) with the
30 g glucose at 15 hours and then both materials again at 23 hours.
The resulting rate of alanine production was consistently above 2.0
g/liter-hour between 15 hours and 27 hours (FIG. 8), significantly
greater than previously when less NH.sub.4Cl was added. In
duplicate experiments, the alanine concentration reached 32 g/liter
in 27 hour, but the alanine concentration did not increase further
regardless of whether additional glucose and NH.sub.4Cl was added.
The overall alanine yield on glucose averaged 0.63 g/g, and the
alanine yield on glucose after 15 hours averaged 0.81 g/g.
Example XI
Reduction of Pyruvate Oxidase Activity
An enzyme that can assimilate pyruvate is pyruvate oxidase. We
observed significant pyruvate oxidase activity (over 1.00 IU/mg
protein) after acetate was depleted in all the fed-batch
fermentations operated at various levels of pH (Example V). These
results suggest that reducing or eliminating pyruvate oxidase
activity would prevent a portion of the pyruvate generated from
being lost.
One approach is to knock out the poxB gene in E. coli expressing
pyruvate oxidase. These strains are expected to grow and accumulate
pyruvate at higher levels under the previously tested
conditions.
The complete disclosures of all patents, patent applications
including provisional patent applications, and publications, and
electronically available material (e.g., GenBank and Protein Data
Bank amino acid and nucleotide sequence submissions) cited herein
are incorporated by reference. The foregoing detailed description
and examples have been provided for clarity of understanding only.
No unnecessary limitations are to be understood therefrom. The
invention is not limited to the exact details shown and described;
many variations will be apparent to one skilled in the art and are
intended to be included within the invention defined by the
claims.
* * * * *